Rolling circle replication of padlock probes

ABSTRACT

Rolling circle replication of a padlock primer is inhibited when it is hybridised to a target nucleic acid that is long or circular. The invention provides methods of addressing this problem including cutting the target nucleic acid near or preferably at the site which hybridises with the padlock probe, whereby a 3′-end of the cut target nucleic acid acts as a primer for rolling circle replication of the padlock probe. Also included is a method for assaying for a polyepitopic target by the use of two affinity probes each carrying an oligonucleotide tag and of padlock probe for rolling circle replication in association with the two affinity probes.

INTRODUCTION

Various methods are in use for detecting single or multiple nucleotidevariations in DNA or RNA samples (1,2). Most methods depend onamplification of the target sequence prior to analysis, commonly by PCR.Thereby, valuable information on the localisation of allelic variants islost. Information on the localisation of sequence variants is importante.g. to determine haplotypes of several variable sequences alongchromosomes in the study of inherited disorders; to determine if twomutations in a gene are present in the same copy or in alternatealleles; or to study replication timing of alleles in the cell cycle (3)or the distribution of mutant cells in a malignant tissue. Singlenucleotide discrimination in single copy gene sequences in situ ispresently not possible, but it has recently been shown to workefficiently on repeated sequences using padlock probes (4), a new classof gene diagnostic probe molecules (5). These are linearoligonucleotides with target complementary sequences at the ends and anon-target complementary sequence in between. When hybridised to thecorrect target DNA sequence, the two ends are brought together, head totail, and can be joined by a DNA ligase. As a consequence of the helicalnature of double stranded DNA the resulting circular probe molecule iscatenated to the target DNA strand. This probe design has some importantfeatures. First, the requirement for two independent oligonucleotidehybridisation events in order for ligation to occur provides sufficientspecificity to detect single-copy genes in the complexity of a completehuman genome (6-48). Second, ligation is greatly inhibited by mismatchesat the ligation junction, allowing single nucleotide distinction in thetarget sequence (4,8-10). Third as the topological link between probeand target DNA strands is independent of hybridisation stability,denaturing washes can be applied reducing non-specific hybridisation(5). Fourth, unlike PCR or LCR, only intramolecular interactions probereactions are scored, avoiding problems of simultaneously applying largesets of probes (11). Whereas combinations of many pairs of PCR primersleads to a rapidly increasing risk of spurious amplification products,formed between any combinations of primers, this is not the case forpadlock probes. Lastly, the joining of the probe ends creates a newclass of molecules, not present before the reaction; these circularmolecules can be amplified in a rolling circle replication reaction todetect ligated probes (12,13).

Amplification of a circular nucleic acid molecule free in solution by arolling circle replication reaction may be achieved simply byhybridising a primer to the circular nucleic acid and providing a supplyof nucleotides and a polymerase enzyme. However, problems may arise whenthe circular nucleic acid is not free in solution. In the particularcase of a padlock primer which is catenated to its target, it mightreasonably be expected that the target would inhibit a rolling circlereplication reaction. In previously unpublished work leading up to thepresent invention, the inventors have demonstrated that such inhibitiondoes indeed take place (18). They found that, if the target is circular,for example the circular 7 kbM13 genome, then rolling circle replicationof a padlock primer catenated to the target is effectively prevented.Where a target is linear, it is possible in principle for a padlockformed thereon to slide along the target molecule and off the end,thereby becoming free in solution and available for amplification by arolling circle replication reaction. Alternatively, the target sequence,3′ of the site of binding by the padlock probe, may be digested byexonucleolysis, allowing the remaining target strand to prime rollingcircle replication. In practice, the inventors have found that thissliding and uncoupling effect is possible to a limited extent even withlong linear targets. Thus, converting the circular 7 kbM13 genome byrestriction at a single site into a linear 7 kb nucleic acid moleculewith 3.5 kb upstream and 3.5 kb downstream of the hybridisation site ofthe padlock primer, limited amplification of the padlock primer waspossible by rolling circle replication. But where the target nucleicacid is substantially shorter, e.g. a few tens or hundred of bases,rolling circle replication of a padlock primer formed thereon is muchmore rapid and efficient.

THE INVENTION

In one aspect of the present invention, this problem is addressed bycutting the target molecule before, during or after target recognitionand probe circularisation. In this aspect the invention provides amethod of detecting a target sequence of a target nucleic acid whichmethod comprises the steps of:

-   i) providing a padlock probe for the target sequence,-   ii) forming a hybrid of the padlock probe with the target nucleic    acid, and circularising the padlock probe,-   iii) cutting the target nucleic acid at or near the target sequence,    this step iii) being performed before, during or after step ii), and-   iv) effecting rolling circle replication of the padlock probe.

The target nucleic acid may in principle be DNA or RNA. The method ofthe invention is likely to be useful particularly when the targetnucleic acid is a long-chain or circular molecule. The target sequenceis of sufficient length to hybridise with the two ends of the padlockprobe. The method of the invention is expected to be particularly usefulfor localised detection of single-copy gene sequences and fordistinction among sequence variants in microscopic specimens, and forprobes immobilised in one or two dimensional arrays.

The padlock probe has a 5′-end sequence and 3′-end sequencecomplementary to the target sequence; so that it can be circularisede.g. by ligation when hybridised to the target sequence. Precisecomplementarity is not necessarily required, but there must besufficient complementarity to permit hybridisation of the padlock probeto the target sequence, and circularisation of the padlock probe byligation. It is in principle possible to provide a padlock probe in twoparts, in which case two ligation reactions are required to achievecircularisation. The size of the padlock probe needs to be suitable topermit amplification by a rolling circle replication reaction,preferably at least about 25 nt and not more than 150-200 nt, thoughlarger probes are possible particularly when used with a polymerase nothaving exonuclease activity, especially 5′ to 3′ exonuclease activity.

Step ii) of the method involves forming a hybrid of the padlock probewith the target sequence of the target nucleic acid, and circularisingthe padlock probe. These steps may be carried out under conventionalconditions. If the target nucleic acid sample under investigation doesnot contain the target sequence, then hybridisation and circularisationof the padlock probe occur not at all or only to a limited extent.

In step iii) of the method the target nucleic acid is cut at or near thetarget sequence. This cut may be made by means well known in the art.Although the distance of the restriction site from the target sequencemay be substantial, e.g. 3.5 kb as above or even greater, it ispreferably within a few bases or a few tens of bases of the targetsequence.

Thus the target nucleic add may be linearised by restriction digestionbefore the addition of a padlock probe. Alternatively, a primerextension reaction may be performed to generate a relatively shortnucleic acid molecule suitable as a target for padlock proberecognition. For in situ detection in metaphase chromosomes, these twomethods are subject to the disadvantage that they risk causing loss oftarget DNA and thereby loss of detection. Preferably therefore step iii)is performed by subjecting the hybrid to restriction thereby cutting thetarget nucleic acid at or near the target sequence but without cuttingthe circularised padlock probe.

In step iv), the padlock probe is amplified by a rolling circlereplication reaction. This usually requires a primer to hybridise to thecircularised padlock probe, a supply of nucleotides and a polymeraseenzyme, and may be effected by means well known in the art. A preferredpolymerase enzyme is φ29 DNA polymerase which has high processivity and3′-exonuclease activity.

Therefore when a target sequence is cleaved 3′ of where a padlock probehas bound, then any non-basepaired nucleotides may be removed by thepolymerase, until a 3′ end basepaired to the padlock probe is obtained,whereupon rolling circle replication (RCR) can be initiated without theaddition of an external primer, and ensuring that the RCR product iscontinuous with the target sequence. Such cleavage can be accomplishedby hybridising an oligonucleotide to the target sequence downstream ofwhere the probe has bound. The sequence can then be cleaved using arestriction enzyme whose recognition sequence has been rendered doublestranded by this hybridisation. Alternatively, a hairpin oligonucleotidecan be used, having one double stranded end containing a recognitionsequence for a type IIS enzyme, e.g. FokI, and another end hybridisingto the target sequence 3′ of where the padlock probe has bound.

If the padlock probe hybridises to the target sequence across arestriction site, then the padlock can be protected e.g. by beingmodified with phosphorothioates, allowing the target sequence to becleaved without opening the padlock.

If the target nucleic acid is RNA, and is restricted at a site nowwithin the target sequence, then a 3′-end capable of initiating rollingcircle replication of the padlock primer can be obtained by RNase Hdigestion.

According to a preferred method, in step iii) the target nucleic acid iscut within the target sequence. An advantage of this method is that the3′-end of the target sequence then constitutes a primer by means ofwhich rolling circle replication of the padlock probe may be effected instep iv). Thereby the rolling circle replication product formed iscontiguous with the target sequence.

To achieve this, use is preferably made of a type IIS enzyme. (Type IISenzymes are sometimes referred to as class IIS enzymes or Type IVenzymes.). Type IIS restriction endonucleases are described in theliterature (14,15). These enzymes interact with two discrete sites ondouble-stranded DNA, a recognition site which is 4-7 bp long, and acleavage site usually 1-20 bp away from the recognition site. One suchtype IIS enzyme is the FokI restriction endonuclease (16,17) whichrecognises a double-stranded 5′-GGATG-3′ site and cuts at the 9^(th) and13^(th) nucleotides downstream from the 5′-3′ and 3′-5′ strandsrespectively. When using FokI with ssDNA and oligonucleotides, cuttingis observed at the 9^(th) or 13^(th) nucleotide from the recognitionsite. Cutting occurs independent of whether a double stranded regionbetween the recognition and cut sites is perfectly complementary or not.

To use a type IIS enzyme in the above method, a specially designedpadlock probe is required, and this forms another aspect of theinvention. In this aspect there is provided an oligonucleotide suitablefor use as a padlock probe for a target nucleic acid sequence, whicholigonucleotide has 5′-end and 3′-end sequences complementary to thetarget sequence; a first site for recognition by a type IIS enzyme; anda second site where at least one nucleotide residue and/orinternucleotide bond is modified to protect the oligonucleotide fromrestriction by the type IIS enzyme.

The first site for recognition by the type IIS enzyme needs to bepositioned on the padlock probe so that the enzyme cuts the targetnucleic acid sequence. And the second site needs to be positioned on thepadlock probe in relation to the first site so as to protect the padlockprobe from being cut by the type IIS enzyme. The relative positions ofthe first and second sites, in relation to the 5′-end and 3′-endsequences, depend on the particular type IIS enzyme to be used. Themodification at the second site may also depend on the particular typeIIS enzyme to be used. For example, when a FokI enzyme is to be used,and similarly for the type II enzyme HincII, phosphorothioateinternucleotide bonds are effective to prevent cleavage of the padlockprobe.

In the method of the invention, the padlock probe is hybridised to thetarget sequence and circularised. Then the padlock probe is madedouble-stranded at the first site for recognition by a type IIS enzyme.Then the enzyme is added and used to cleave the target sequence. Thenconditions are adjusted to so as to cause a cleaved fragment of thetarget sequence to act as a primer to initiate rolling circlereplication of the padlock probe. Either (or neither) of the padlockprobe and the target nucleic acid may be immobilised.

The system is illustrated diagrammatically in FIG. 1 of the accompanyingdrawings. This shows that in the first stage, a target nucleic acid 10has been immobilised on a support 12. A padlock probe 14 has beenhybridised to a target sequence of the target nucleic acid and has beencircularised. An oligonucleotide primer 16 has been hybridised to afirst site of the padlock probe for recognition by a type IIS enzyme 18,which is about to cut the target nucleic acid within the targetsequence. In the second stage of the figure, the type IIS restrictionendonuclease has cut the target sequence at 20 and the resulting 3′-end22 has been chain extended by a rolling circle replication reactioninvolving the padlock probe 14.

The alternative in which the target nucleic acid is cut beforehybridisatlon with a probe, gives rise to another possibility. This isthat the target nucleic acid fragment that results from cutting mayitself be circularised and amplified by rolling circle replication. Thusin this aspect the invention provides a method of detecting a targetnucleic acid having two non-adjacent target sequences, which methodcomprises the steps of:

-   i) Cutting the target nucleic acid so as to create a target nucleic    acid fragment having a 5′-end target sequence and a 3′-end target    sequence,-   ii) Providing a probe having two adjacent sequences complementary to    the target sequences,-   iii) Forming a hybrid of the target nucleic acid fragment with the    probe, and circularising the target nucleic acid fragment, and-   iv) Effecting rolling circle replication of the circularised target    nucleic acid fragment. Preferably this step is performed using the    probe as primer.

If the rolling circle product is digested and the size of the monomersestimated by gel electrophoresis, then the size of the target sequencecan be estimated. This can be of value to estimate the size oftrinucleotide repeats in conditions such as the fragile X syndrome.Alternatively, the circularised molecule or rolling circle replicationproducts thereof can be investigated, e.g. by DNA sequence analysis. Ina variant of the above, both the probe and the target nucleic acid canbe designed to undergo circularisation. In this latter case, either canbe opened to prime rolling circle replication of the other.

This aspect of the invention is illustrated in FIG. 4 of theaccompanying drawing, in which a target nucleic acid fragment 48 insolution has hybridised to an immobilised probe 50, and has beencircularised. Thereafter, the immobilised probe 50 will act as a primerfor rolling circle replication of the target nucleic acid fragment 48.

In another alternative it is possible, and perhaps advantageous, to usetwo (or more) padlock probes instead of just one. In this aspect of theinvention there is provided a method of detecting a target sequence of atarget nucleic acid, which method comprises the steps of:

-   i) Providing a first padlock probe having 5′-end and 3′-end    sequences which are complementary to the target sequence, and an    intermediate sequence,-   ii) Providing a second padlock probe having 5′-end and 3′-end    sequences which are complementary to the intermediate sequence of    the first padlock probe,-   iii) Forming a hybrid by hybridising the first padlock probe to the    target nucleic acid and hybridising the second padlock probe to the    first padlock probe, and circularising both padlock probes,-   iv) Purifying the hybrid,-   v) Subjecting the hybrid to restriction thereby cutting the first    padlock probe, and-   vi) Effecting rolling circle replication of the second padlock    probe.

In step iii), a hybrid is formed by hybridising the first padlock probeto the target nucleic acid and hybridising the second padlock probe tothe first padlock probe, and circularising both padlock probes. Thetechniques for performing these steps are well known in the art and maybe as discussed above. Any one (or none) of the target nucleic acid, thefirst padlock probe or the second padlock probe may be immobilised.

Step iv) of the method involves purifying the hybrid, which may be doneby means of superstringent washing, e.g. using pH, temperature orpercentage formamide to denature the DNA hybrid. This step is effectiveto remove: any second padlock probe that has not been circularised; andany second padlock probe that has hybridised to a first padlock probewhich has itself not been circularised (e.g. because it has nothybridised to a target sequence). This separation may be aided if eitherthe target nucleic acid or the first padlock probe is immobilised on asolid support. In step v), the first padlock probe is cut, and in stepvi) rolling circle replication of the second padlock probe is effected.As in the previous method, it is preferred that in step v) the firstpadlock probe is cut within the intermediate sequence which ishybridised to the second padlock probe, to provide a primer by means ofwhich rolling circle replication of the second padlock probe is effectedin step vi).

Cutting the first padlock probe within its intermediate sequence may beeffected by the use of a type IIS restriction endonuclease as describedabove. Alternatively, the intermediate sequence of the first padlockprobe may include modified residues that may be cleaved, such asdU-residues, susceptible to base removal by UTP glycosylase, followed bycleavage by ExoIII or alkali or heat. Since the intermediate sequencemay be chosen at will, it may be designed to include a recognitionsequence of any convenient restriction enzyme e.g. HincII, with thesecond padlock probe being protected e.g. by provision ofphosphorothioate linkages.

The system is illustrated in part A of FIG. 2. In the first part of thisdiagram, a hybrid has been formed by hybridising to a first padlockprobe 24, which is immobilised on a solid support 26, a target sequencein solution 28, and a second padlock probe 30. Both padlock probes havebeen circularised. At the second stage, the first padlock probe has beencut at 32 (without cutting the second padlock probe), and the 3′-end ofthe restriction fragment 34 has acted as a primer for rolling circlereplication of the second padlock probe.

Also included within the scope of this invention is a kit for performingthe above method, which kit comprises the first padlock probe and thesecond padlock probe as described. In this and other cases, it willgenerally be convenient for the nucleotides used for rolling circlereplication to include a labelled nucleotide for easy detection.Alternatively, the rolling circle replication product can beinvestigated using a hybridisation probe.

In FIG. 2B, a target nucleic acid fragment 27 has hybridised to thepadlock probe 25. The padlock probe has been circularised and is aboutto be amplified by rolling circle replication using an immobilisedoligonucleotide 29 as primer. In a development of this technique, boththe padlock probe 25 and the target nucleic acid fragment 27 can becircularised and thus catenated if the 5′- and 3′-ends of each moleculecan hybridise to the other, but in such a way that the ends to be joinedby ligation do not hybridise immediately opposite one another. Thisprobe-target configuration offers increased specificity of detection;but rolling circle replication of one molecule requires linearisation ofthe other, preferably within the base-paired region.

Patent specification WO 96/14406 describes enzymatic synthesis ofpadlock probes. These probes can also be detected via a rolling circlemechanism if catenation can be avoided. This can be achieved if they arefirst generated with a type IIS restriction enzyme and the 5′-phosphatereplaced with a thiophosphate by phosphatase treatment, followed bykinasing with γ-thiophosphate ATP. In this manner these probes are alsoprotected from digestion with the same IIS enzyme after targetrecognition as described above. Binding of such enzymatically preparedprobes can also be visualised using a linked detection padlock probe asalso described above.

According to patent specification WO 97/00446. enhanced immune detectioncan be achieved by requiring coincident binding of two or more affinityprobes, for example antibodies, to a target molecule. Upon targetrecognition, oligonucleotides bound to the affinity probes are broughtclose enough to be joined by ligation forming a longer oligonucleotidewhich can template an expotential nucleic acid amplification reactionthrough PCR or NASBA, etc. In this aspect, the present invention uses arelated approach but employing rolling circle replication of a padlockprobe. Thus the invention provides a method of assaying for apolyepitopic target by providing:

-   a) A first affinity probe for the target which first affinity probe    carries a polynucleotide chain including a first polynucleotide    sequence,-   b) A second affinity probe for the target which second affinity    probe carries a polynucleotide chain including a terminal second    polynucleotide sequence, and-   c) A padlock probe having 5′-end and 3′-end sequences which are    complementary to the first polynucleotide sequence, and an    intermediate sequence which is complementary to a 3′-end of the    second polynucleotide sequence,

which method comprises binding the first affinity probe to the target;binding the second affinity probe to the target; hybridising the padlockprobe to the first polynucleotide sequence and to the secondpolynucleotide sequence; circularising the padlock probe; and using thesecond polynucleotide sequence as a primer to effect rolling circleamplification of the padlock probe.

The invention also provides a kit for performing the method, which kitcomprises the first and second affinity probes and the padlock probe.Preferably the first affinity probe and the second affinity probe areselected from: polyclonal, monoclonal and single chain antibodies andfragments thereof; receptors, lectins and nucleic acid aptamers.

In the method, the first affinity probe and the second affinity probeare incubated with the target, under conditions to cause them to bind tothe target. Then the padlock probe is hybridised to the firstpolynucleotide sequence and is circularised. If the two affinity probesbound to the target are close enough together, then the terminal secondpolynucleotide sequence of the second affinity probe becomes hybridisedto the intermediate sequence of the padlock probe. On addition of asupply of nucleotides and a polymerase enzyme, rolling circlereplication of the padlock probe can be effected. Use of a radioactivelyor otherwise labelled nucleotide provides a correspondingly amplifiedsignal related to the presence or concentration of the polyepitopictarget.

The system is illustrated in FIG. 3 of the accompanying drawings, inwhich there is shown a polyepitoplc target 36, a first antibody 38 forthe target which carries a polynucleotide chain 40 including a firstpolynucleotide sequence; a second antibody 42 for the target whichcarries a polynucleotide chain 44 including a terminal secondpolynucleotide sequence; and a padlock probe 46 which has hybridised toboth polynucleotide chains and has been circularised. Upon addition of asupply of nucleotides and a polymerase enzyme, the polynucleotide chain44 will act as a primer for rolling circle replication of the padlockprobe 46.

Rolling circle products generated by any of the methods described hereincan be visualised during synthesis using so called molecular beacons. (STyagi and F R Kramer, 1996. Nature Biotechnology, 14, 303-308). Amolecular beacon is a usually hairpin shaped oligonucleotide carrying afluorescing label at one end, and at the other end a compound thatmodulates or inhibits the fluorescence. Unfolding the normallyhairpin-shaped molecular beacon modulates or enhances the fluorescencesignal in an easily observed way. A molecular beacon designed to have asequence corresponding to that of a padlock probe, can be used tomonitor rolling circle replication of the padlock probe.

This system is illustrated in FIG. 5 of the accompanying drawings. Apadlock probe 52 has been hybridised to a target sequence of a targetnucleic acid 54 and has been circularised. The target nucleic acid hasbeen cut at 56 and the resulting 3′-end 58 has been chain extended by arolling circle replication reaction involving the padlock probe 52. Amolecular beacon 60 has a terminal fluorescent group 62 and a terminalquenching group 64, and an intermediate sequence corresponding to thatof the padlock probe 52. As rolling circle replication takes place,successive molecules of the molecular beacon become hybridised to theextending chain at 66 in a conformation which permits enhancedfluorescence of the fluorescent group.

Reference is directed to FIGS. 6-14, which are described in thefollowing.

EXPERIMENTAL SECTION

Materials and Method

Oligonucleotides synthesised by Interactiva:

-   M13-Fo1s (56 nt) contains 6 phosphorothioates-   M13-Fo2 (56 nt) contains no phosphorothioates-   M13-Ls1 (23 nt) contains FokI cleavage-site-   M13-T3s is used as template-   M13-K1 (34 nt) is used as a FokI cleavage adapter.    Oligonucleotides synthesised in house using an ABI 394 DNA    Synthesiser:-   M13c70-roI (70 nt) used as a padlock probe-   Primer MvaI (21 nt) used for M13 cleavage by MvaI.-   RvRoIcpr (21 nt) used for cleavage of RCR-product by HpaII    Labelling:

The probes were 5′-radiolabelled using 30 units T4 polynucleotide kinase(Amersham) in 50 μl 10 mM Tris Ac pH 7.5, 10 mM MgAc₂, 50 mM KAc and 30μCi gamma 32P-ATP (3000 mCi/mMol, NEN Dupont) at 37° C. for 10 min, andthe oligonucieotides were purified on a Sephadex G50 μspin column(Pharmacia).

FokI cleavage using oligonudeotides:

Oligonucleotides, one labelled at the time, were mixed and allowed tohybridise by incubating 1 pmol M13-T3S, 2 pmol M13-Fo1s or M13-Fo2together with 3 pmol M13-Ls1 or M13-K1 in 20 μl 10 mM TrisAc pH 7.5, 10mM MgAc₂, 50 mM KAc, 1 mM ATP, 0.1 μg/μl BSA at 65° C. for 10 min andallowed to cool at room temperature for 10 min. FokI was added to a concof 0.5 units/μl and the reaction was incubated at 37° C. for 60 min. Theenzyme was heat inactivated at 65° C. for 20 min and the reaction wasallowed to cool to room temperature for 10 min.

FokI cleavages of M13 using padlock and FokI-adapter:

0.75 pmol M13mp18+strand (Pharmacia), 0.25 pmol M13c70-RoI (labelled)and 1.5 pmol M13-K1 were mixed and allowed to hybridise as describedabove. The FokI cleaved was peformed as described above.

MvaI cleavages of M13 using padlock and MvaI-primer:

0.75 pmol M13mp18+strand (Pharmacia), 0.25 pmol M13c70-RoI (labelled)and 1.5 pmol MvaI-primer in 20 μl 10 mM TrisAc pH 7.5, 10 mM MgAc₂, 50mM KAc, 1 mM ATP, 0.2 μg/μl BSA was incubated at 65° C. for 10 min andallowed to cool at room temperature for 10 min. 2 units MvaI was addedand the reaction was incubated at 37° C. for 60 min. the enzyme washeat-inactivated at 65° C. for 15 min and the reaction was allowed tocool to room temperature for 10 min.

Phi 29 DNA polymerase reaction:

1 μl enzyme reaction was added to 50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 20mM (NH₄)₂SO₄, 1 mM dithiothreitol and 0.2 μg/μl BSA, 0.25 mM dNTP, 5 μCialpha 32P dCTP (3000 Ci/mmol, NEN Dupont) and 2 ng/μl Phi29 DNApolymerase (provided from Amersham) and incubated at 37° C. for 60 min.The enzyme was heat inactivated at 65° C. for 20 min.

Cleavages of RCR-product:

1 μl RCR-reaction is added in 10 mM Bis Tris Propane-HCl, 10 mM MgCl₂, 1mM dithiothreitol (pH 7.0) and 1 pmol/μl RvRoIcpr and is incubated at65° C. for 10 min and allowed to cool at room temperature for 10 min. 1units HpaII was added and the reaction was incubated at 37° C.overnight. The enzyme was heat inactivated at 65° C. for 20 min.

EXAMPLE 1 Strand Specific Cleavage by FokI and Protection byPhosphorothioates (FIG. 6).

Enzyme titration. This experiment involves two partially complementaryoligonucleotides, 29 and 39 nucleotides in length. Both were labelled atthe 5′ end by kinaseing, adding a 32P residue. In the left hand set 1through 8, both oligonucleotides were composed of regular nucleotides,and addition of the type IIS restriction endonuclease FokI, resulted incleavage of both strands. This results in loss of the 29-mer, and gainof a 17-mer fragment. Similarly, the 39-mer is replaced by an 8-mer. Inthe right-hand set 1 through 8, the 29-mer contained aphosphorothioate-residue where the restriction enzyme otherwise wouldhave cleaved. The experiment shows that phosphorothioate-residues blockcleavage of the modified strand without inhibiting cleavage of the otherstrand. (This property has not been previously demonstrated for thisenzyme, but it is well established for several type II enzymes, havingcleavage sites within their recognition sequence.)

EXAMPLE 2 Strand-Specific Cleavage Using FokI Followed by Phi29Polymerisation (FIG. 7)

Here a branched substrate for restriction digestion was used.

The 56-mer is complementary to a 23-mer at its 5′-end and to a(radiolabelled) 71-mer at its 3′ end. The 23-mer includes therecognition sequence of the enzyme FokI, but is cleavage site is locatedacross the branch. The sample shown in lane 3 is untreated. The samplein lane 1 was treated with the restriction enzyme, generating a shorterfragment of 33. In lane 4 the polymerase Phi29 and nucleotides had beenpresent, with no consequences, compared to lane 3, as expected. Finally,in lane 2 restriction was followed by polymerisation. Only a smallfraction of the cleavage products have been extended by a fewnucleotides to fill in the 5′ overhang generated by restrictioncleavage. This experiment demonstrates that cleavage across a branch ispossible, as has been previously shown by Szybalski. The reason for thepoor polymerisation in this experiment, as required to initiate arolling circle replication, is not clear. (The 100 nt fragment is anunintended extension product of the 71-mer.).

EXAMPLE 3 Strand-Specific Cleavage Using FokI and Protection byPhosphorothicates Followed by Phi29 Polymerisation (FIG. 8)

The experiment is similar to the previous one, except that in thisexperiment the 56-mer was protected from restriction cleavage by beingmodified with phosphorothioates. Lane 3: starting material. Lane 1:restriction cleaved. Lane 4: Addition of polymerase and nucleotides;most 56-mers remain unchanged (, although some seem to have beenextended somehow as mentioned for the above experiment). Lane 2: Heremost of the 33-mer cleavage products have been extended, copying the nowuncleaved 56-mer, just as would be required to initiate a rolling circlereplication by directing a cleavage reaction to the target strand forpadlock recognition, allowing the target to prime a rolling-circlereplication reaction.

EXAMPLE 4 Strand-Specific Cleavage by FokI and Protection byPhosphorothioates (FIG. 9)

A simpler variant of the preceding experiment, but with the 56-merstrand 5′ labelled with radioactive phosphate. The unprotected strand iscleaved as expected, but the protected one is cleaved farther away thannormal, where it is unprotected, indicating that a larger sequence mayhave to be protected by being modified with phosphorothioates.

EXAMPLE 5 Cleavage by FokI by Using a FokI-Adapter (FIG. 10)

In this experiment an alternative means is used to cleave thepadlock-target strand, in this case downstream of where the probe hasbound. An oligonucleotide that includes a self-complementary segmentdirects recognition by the restriction enzyme and cleavage where thisadapter hybridises to a target strand. Lane 3: no restriction enzyme.Lane 1: restriction resulting in a shorter fragment of 43 bases. Lane 4:no effect by addition of polymerase. Lane 2, some of the 43-mer cleavageproduct is extended by the polymerase to generate a 56-mer, templated bythe unlabelled 56-mer oligonucleotide.

EXAMPLE 6 Initiation of Rolling Circle Replication by Cleavage of theTarget Strand (FIG. 11)

In this experiment the external adapter of the previous experiment wasused to cleave the circular single stranded M13 target for recognitionby a padlock probe. Labelling was by incorporation of radioactivenucleotides, and with a 5′-labelled padlock probe. This experiment asshown here is not conclusive because unligated padlocks can prime arolling-circle replication, templated by M13. This is addressed in thegel shown in Example 8.

EXAMPLE 7 Initiation of Rolling Circle Replication by Cleavage of theTarget Strand (FIG. 12)

Similar to the preceding experiment except that cleavage of the targetstrand took advantage of a resident restriction site which was rendereddouble stranded by hybridising an ollgonucleotide. Again the two typesof polymerised molecules are distinguished in Example 8.

EXAMPLE 8 Cleavage of Rolling Circle Product With Restriction Enzyme(FIG. 13)

Here the experiments shown in Examples 6 and 7 have been furtherinvestigated, to reveal that the polymerase Phi29 can effect arolling-circle replication of the circularised padlock probes, and thatthe reaction was primed by the target sequence after the target M13strand was cleaved using a FokI adapter (Example 6) or the residentrecognition sequence for MvaI. Ligase indicates whether the padlockprobe has been ligated. RE means either FokI (lanes 1-8) or MvaI (lanes9-16), used as a means to cleave the target molecule downstream of thesite where the padlock probe has bound. All samples were treated withPhi29 polymerase. Products of the extension reaction shown ineven-number lanes were treated with the restriction enzyme HpaII in thepresence of oligonucleotide RvRo1cpr to cleave the rolling-circleproducts to monomers. The cleavage allowed identification ofpolymerisation products that arise by copying of the circularisedpadlock probe, as opposed to ones templated by the M13 genome. The70-mer product of the enzyme reaction which is visible in lanes 8 and 16clearly shows that a rolling circle replication was primed from thetarget strand by cleaving the M13 molecule that served as target forligation, downstream of where the padlock probe had bound. This reactiongenerated copies of the padlock probe.

FIG. 14 shows the four oligomers M13-Fo1s (56 nt); M13-Ls1 (23 nt);M13-K1 (34 nt) and M13-T3s (71 nt) hybridised together. Sites for Fok1binding and cleavage, and phosphorothioate bonds, are shown togetherwith oligonucleotide lengths.

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1. (canceled)
 2. A method comprising: providing a first affinity probefor a target which first affinity probe carries a polynucleotide chainincluding a first polynucleotide sequence, a second affinity probe forthe target which second affinity probe carries a polynucleotide chainincluding a second polynucleotide sequence, a padlock probe provided intwo parts wherein the first part of the padlock probe has a 5′ end whichis complimentary to the first polyoligonucleotide and a 3′ endcomplementary to the second polyoligonucleotide, and the second part ofthe padlock probe has a 3′ end which is complementary to the firstpolyoligonucleotide and a 5′ end complementary to the secondpolyoligonucleotide; binding the first affinity probe to the target;binding the second affinity probe to the target; hybridizing the padlockprobe provided in two parts; circularizing the padlock probe; andeffecting amplification of the padlock probe.
 3. The method of claim 2,wherein the amplification is effected by rolling-circle amplification.4. The method of claim 2, wherein the first or the second polynucleotidesequence is used as primer for the amplification.
 5. The method of claim2, wherein amplification is effected using Phi29 DNA polymerase.
 6. Akit for assaying for a polyepitopic target which comprises: a) a firstaffinity probe for the target which first affinity probe carries apolynucleotide chain including a first polynucleotide sequence, b) asecond affinity probe for the target which second affinity probe carriesa polynucleotide chain including a second polynucleotide sequence, andc) a padlock probe provided in two parts wherein the first part of thepadlock probe has a 5′ end which is complementary to the firstpolyoligonucleotide and a 3′ end complementary to the secondpolyoligonucleotide, and the second part of the padlock probe has a 3′end which is complementary to the first polyoligonucleotide and a 5′ endcomplementary to the second polyoligonucleotide.
 7. (canceled)
 8. Amethod comprising: providing a first affinity probe for a target whichfirst affinity probe carries a polynucleotide chain including a firstpolynucleotide sequence, a second affinity probe for the target whichsecond affinity probe carries a polynucleotide chain including a secondpolynucleotide sequence, two or more padlock probes, each padlock probebeing provided in two parts, wherein a first padlock probe has 5′ endand 3′ end sequences that are complementary to the target sequence, andan intermediate sequence, and a second padlock probe has 5′ end and 3′end sequences which are complementary to the intermediate sequence ofthe first padlock probe, binding the first affinity probe to the target;binding the second affinity probe to the target; hybridizing the firstpadlock probe to the target sequence and hybridizing the second padlockprobe to the first padlock probe; circularizing the padlock probes; andeffecting amplification of the padlock probes.
 9. The method of claim 8,wherein the amplification is effected by rolling-circle amplification.10. The method of claim 8, wherein the first or the secondpolynucleotide sequence is used as primer for the amplification.
 11. Themethod of claim 8, wherein amplification is effected using Phi29 DNApolymerase.
 12. A kit for assaying for a polyepitopic target whichcomprises: a) a first affinity probe for the target which first affinityprobe carries a polynucleotide chain including a first polynucleotidesequence, b) a second affinity probe for the target which secondaffinity probe carries a polynucleotide chain including a secondpolynucleotide sequence, and c) two or more padlock probes, each padlockprobe being provided in two parts, wherein a first padlock probe has 5′end and 3′ end sequences which are complementary to the firstpolynucleotide sequence, and an intermediate sequence, and a secondpadlock probe has 5′ end and 3′ end sequences which are complementary tothe intermediate sequence of the first padlock probe and the secondpolynucleotide sequence.